Project Summary RNA polymerase (RNAP) pausing and termination are important components of gene expression in all organisms. NusA and NusG are two general transcription elongation factors that are capable of stimulating pausing and termination in bacteria. Pausing allows synchronization RNAP position with RNA folding and/or regulatory factor binding. Intrinsic and Rho-dependent termination are two transcription termination mechanisms identified in bacteria. Canonical intrinsic terminators consist of an uninterrupted RNA hairpin followed by a U-tract. Although NusA was known to stimulate intrinsic termination in vitro, since NusA is essential for viability its role on termination in vivo was not known until recently. RNA-seq studies with a B. subtilis NusA depletion strain identified a class of intrinsic terminator that requires NusA. NusA-dependent terminators have weak RNA hairpins and/or poor U-tracts. There is also evidence that NusG stimulates termination of mycobacterial RNAP at suboptimal intrinsic terminators in vitro. In Rho-dependent termination, Rho promotes transcript release when it catches up to paused RNAP. E. coli NusG participates in some Rho- dependent termination events by serving as a bridge between RNAP and Rho. NusA and NusG cooperatively stimulate pausing at two sites in the 5'UTR of the B. subtilis trp operon. NusG makes sequence-specific contacts with the non-template DNA (ntDNA) strand within the paused transcription bubble. As RNAP and template DNA must move with respect to one another for elongation to resume, interaction of NusG with both components inhibits elongation. The T-rich ntDNA sequence at the two pause sites constitutes a conserved NusG recognition motif. NET-seq will be used to identify pause sites throughout the B. subtilis genome that respond to NusA and/or NusG. The ability to deplete NusA and delete nusG without growth defects makes B. subtilis the ideal organism for these studies. By combining RNase footprinting with NET-seq (RNET-seq), the effect of NusA and NusG on the translocation state of RNAP will be determined at each pause site. Similarly, a comprehensive genomic analysis of the effects of NusA, NusG and Rho on termination in B. subtilis will be performed using strains containing all combinations of NusA depletion, ?nusG and ?rho alleles. A subset of regulatory pause sites and terminators will then be characterized in vitro. A hallmark of transcription attenuation mechanisms is the presence of overlapping antiterminator and terminator structures that form in the 5'UTR. The 5'UTR of B. subtilis yxjB contains two such sets of overlapping structures. A model will be tested in which YxjB autoregulates its expression by binding to its 5'UTR and promoting termination at both terminators by preventing formation of the two antiterminators. The model also posits that the downstream terminator hairpin sequesters the yxjB ribosome binding site. Thus, this hairpin would repress translation of transcripts that fail to terminate. A combination of in vivo expression, in vitro transcription and in vitro binding studies will be used to elucidate these complex regulatory mechanisms.